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[Bio101] Cell Culture Transfection for Production and Purification of Wnt Ligands
[Bio101] 培养细胞中Wnt配体的生产和纯化   

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Abstract

Wnt ligand proteins are extremely difficult to purify and enrich in vitro. This protocol uses Wnt11r protein as an example to illustrate how to use 293T cells to produce secreted Wnt11r and collect it in vitro for further biochemical experiments.

Materials and Reagents

  1. 293T cells
  2. 0.1 M glycine-HCl (pH 3.5)
  3. 0.5 M Tris-HCl
  4. Fetal bovine serum (FBS) (Life Technologies, Invitrogen™, catalog number: 10438-026 )
  5. Dulbecco's modified eagle medium (DMEM) (high glucose) (Life Technologies, Invitrogen™, catalog number: 11965 )
  6. Trypsin EDTA (Life Technologies, Invitrogen™, catalog number: 15050-065 )
  7. PBS buffer (Life Technologies, Invitrogen™, catalog number: 14040-182 )
  8. Anti-FLAG M2 (Sigma-Aldrich, catalog number: A2220 )
  9. Pen/Strep (Sigma-Aldrich, catalog number: P4333 )
  10. Effectene transfection regent (QIAGEN, catalog number: 301425 )
  11. Media (see Recipes)

Equipment

  1. Standard tabletop centrifuges
  2. 37 °C, 5% CO2 incubator
  3. Water bath
  4. 50 ml conical tubes
  5. Nitrocellulose membrane
  6. 100 mm Petri dishes

Procedure

  1. Split 293T cells:
    1. Warm media and PBS buffer in 37 °C water bath and 0.05% Trypsin EDTA at RT. Get one 50 ml conical tube, and a 100 mm dish to pass cells.
    2. Remove old medium by aspiration. Add 10 ml PBS buffer to rinse the cells. Gently shake the dish. Remove Hass buffer by aspiration.
    3. Add 1 ml 0.05% Trypsin-EDTA to 100 mm dish confluent with 293T cells. Incubate at 37 °C for 1 min. Gently rock dish. Cells should come off from the dish.
    4. Add 9 ml media and gently pipet up and down to dissociate cells from plate.
    5. Once dissociated, pour into 50 ml conical tube.
    6. Centrifuge cells at 1,000 rpm for 3-4 min and then remove the supernatant with vacuum-attached Pasteur pipet.
    7. Add 10 ml medium and pipet up and down to resusupend cells.
    8. Add 9 ml fresh media and 1 ml of resuspended cells to a fresh dish. This dish will become confluent by day 5.
      Note: You can also do a 1:5 and 1:20 dilution, in which the cells will become confluent at day 3 and day 7 respectively.
    9. Grow cells in 37 °C, 5% CO2 incubator. Check the media 3 days later, if the media turns yellowish, change the media.

  2. Seed the cells and transfection:
    1. Split the cells at 1:10 ratio as steps 1-6 in 100 mm dish. 
    2. Grow cells for 16-24 h. The cells will become 30-40% confluent.
    3. Warm media and Hass buffer at 37 °C. Mix DNA (Wnt11r expression construct with a FLAG tag) in the hood. Follow the transfection protocol from Qiagen:
      1. Mix 8 μg DNA in 300 μl EC buffer.
      2. Add 64 μl enhancer and mix well by pipetting. Incubate in the hood for 5 min. 
      3. Add 60 μl for effectene and mix well and incubate for 10 min.
      4. Add 3,000 μl media to the mix.
        Note: The amounts above are for transfection in one 100 mm dish.
      5. During the incubation, remove the old media from the cells and rinse once by 10 ml PBS buffer.
      6. Add 7 ml fresh media to the cells. When the incubation is over, add the mix (total 3,424 μl: 300 μl+64 μl+60 μl+3,000 μl) to the dish. Gently shake the dish.
      7. Grow the cells in 37 °C, CO2 incubator.
    4. Day 2: Remove transfection media from dish. 
    5. Add another 10 ml fresh media (slowly pipet media along edge; cells will dislodge by addition of media by pipet). Grow for 3-4 days.
      Note: This step is completely optional. The cells grow very well in effectene transfection media.

  3. Collection of Wnt11r-FLAG supernatant:
    1. After 4 days of growth in transfection media collect media in 15 ml conical tube. Spin 5 min at 1,000 rpm.
    2. Filter through a nitrocellulose membrane (0.2 mm). According to literature, wnt ligands can be stored in medium for several months at 4 °C without loss of activity. Not clear if this is true for wnt11r.
      Optional: Aliquot supernatant in 1 ml aliquots and store in -80 °C. 
    3. Pull-down the wnt11r-FLAG proteins by anti-FLAG beads:
      1. Thoroughly susupend the anti-FLAG M2 gel in the vial. The ratio of suspension to packed gel volume is 2:1 (i.e. if you want to get 20 μl packed beads, take 40 μl out from the vial using the wide-end tips).
      2. Clean the anti-FLAG beads following the manufacture’s directions.
      3. Wash the beads 4 times using cold TBS buffer (500 μl for 40 μl of gel suspension).
      4. Centrifuge the beads at 5,000-8,000 x g for 30 sec. Wait for 1-2 min before handling the samples. Remove the supernatant with a narrow-end pipet tip. Be careful not to transfer any beads.
      5. Wash the beads with 0.1 M glycine-HCl pH 3.5 to reduce the traces of unbound anti-FLAG antibody from the suspension
        Note: Do not leave the beads in glycine HCl for longer than 20 min.
      6. Wash the beads again with TBS buffer for total 3 times.
      7. Add wnt11r-containing medium to the beads. Leave the tubes on the wheel at 4 °C O/N.
        Optional: You can add proteinase inhibitors to the medium before adding them to the beads.
        Note: Every step above should be performed on ice or at 4 °C.
    4. Elute the wnt11r-FLAG proteins using glycine-HCl buffer:
      1. Centrifuge the beads and remove the medium with a narrow-end pipet tip.
      2. Wash the beads 3 times with TBS buffer. Make sure all the supernanant is removed.
      3. Elute wnt11r-FLAG proteins with 0.1 M glycine-HCl.
      4. Add 100 μl of 0.1 M glycine-HCl buffer to the beads. Incubate the samples with gentle shaking at RT for 5 min.
      5. Centrifuge at 5,000-8,000 x g for 30 sec.
      6. Transfer the supernatant to a fresh tubes containing 10 μl of 0.5 M Tris-HCl (pH 7.4), 1.5 M NaCl. Be careful not to transfer any beads.
      7. For immediate use, store the supernatant at 4 °C. Store at -20 °C for long-term storage.
    5. Determine the amount of protein in the elution buffer:
      1. Take 10 μl of eluted proteins and add 10 μl 2x SDS sample buffer. Boil for 5 min and then run a SDS-PAGE gel. For markers, run BSA with different amounts in each lane. Stain the gel with G-blue and then determine the amount of protein present in 10 μl elution.
      2. The size of wnt11r-FLAG is 44 kDa.

Recipes

  1. Media
    DMEM
    10%FBS
    1%Pen/Strep

Acknowledgments

This protocol was developed in the Michael Granato Lab at University of Pennsylvania, Philadelphia, USA, and this work was supported by NIH grant R01HD037975.

References

  1. Jing, L., Lefebvre, J. L., Gordon, L. R. and Granato, M. (2009). Wnt signals organize synaptic prepattern and axon guidance through the zebrafish unplugged/MuSK receptor. Neuron 61(5): 721-733.

简介

Wnt配体蛋白在体外非常难以纯化和富集。 该协议使用Wnt11r蛋白作为例子说明如何使用293T细胞产生分泌的Wnt11r,并收集在体外进行进一步的生化实验。

材料和试剂

  1. 293T细胞
  2. 0.1M甘氨酸-HCl(pH3.5)
  3. 0.5M Tris-HCl
  4. 胎牛血清(FBS)(Life Technologies,Invitrogen TM,目录号:10438-026)
  5. Dulbecco's改良的Eagle培养基(DMEM)(高葡萄糖)(Life Technologies,Invitrogen TM,目录号:11965)
  6. 胰蛋白酶EDTA(Life Technologies,Invitrogen TM,目录号:15050-065)
  7. PBS缓冲液(Life Technologies,Invitrogen TM,目录号:14040-182)
  8. 抗FLAG M2(Sigma-Aldrich,目录号:A2220)
  9. Pen/Strep(Sigma-Aldrich,目录号:P4333)
  10. Effectene转染试剂(QIAGEN,目录号:301425)
  11. 媒体(见配方)

设备

  1. 标准台式离心机
  2. 37℃,5%CO 2培养箱
  3. 水浴
  4. 50ml锥形管
  5. 硝酸纤维素膜
  6. 100 mm培养皿

程序

  1. 裂解293T细胞:
    1. 温热培养基和PBS缓冲液在37℃水浴和0.05%胰蛋白酶EDTA在室温。 得到一个50毫升锥形管,和一个100毫米的培养皿通过细胞。
    2. 通过吸气除去旧介质。 加入10毫升PBS缓冲液冲洗细胞。 轻轻摇动菜。 通过抽吸移除Hass缓冲区。
    3. 加入1ml 0.05%胰蛋白酶-EDTA到与293T细胞汇合的100mm培养皿中。 在37℃孵育1分钟。 轻轻地盘菜。 细胞应该从盘子上脱落。
    4. 加入9毫升培养基,轻轻地吸上下来从板解离细胞。
    5. 一旦解离,倒入50ml锥形管中。
    6. 离心细胞在1,000 rpm 3-4分钟,然后用真空附着的巴斯德吸管除去上清液。
    7. 加入10毫升培养基和吸移管向上和向下到resusupend细胞。
    8. 加入9毫升新鲜培养基和1毫升重悬浮的细胞到新鲜的菜。 这道菜将在第5天汇合。
      注意:您还可以进行1:5和1:20稀释,其中细胞在第3天和第7天分别融合。
    9. 在37℃,5%CO 2培养箱中生长细胞。 3天后检查介质,如果介质变黄,更换介质。

  2. 种子细胞和转染:
    1. 在100mm培养皿中以1:10的比例分离细胞,如步骤1-6。
    2. 生长细胞16-24小时。 细胞将变成30-40%汇合。
    3. 温热培养基和Hass缓冲液在37℃。 混合DNA(Wnt11r表达构建与FLAG标签)在敞篷。 按照Qiagen的转染方案:
      1. 混合8微克DNA在300微升EC缓冲液。
      2. 加入64微升增强剂,通过吸移混匀。 在通风橱中孵育5分钟。
      3. 加入60μl的Effectene,混匀,孵育10分钟。
      4. 加入3,000微升培养基的混合物。
        注意:以上数量用于在一个100mm培养皿中进行转染。
      5. 在孵育过程中,从细胞中取出旧培养基,并用10ml PBS缓冲液冲洗一次。
      6. 加入7毫升新鲜培养基的细胞。 当温育结束时,将混合物(总共3,424μl:300μl+64μl+60μl+3,000μl)加入到培养皿中。 轻轻摇动菜。
      7. 在37℃,CO 2培养箱中生长细胞。
    4. 第2天:从盘中取出转染培养基。
    5. 加入另外10毫升新鲜培养基(沿边缘缓慢移液培养基;细胞将通过移液管添加培养基移除)。 生长3-4天。
      注意:此步骤是完全可选的。 细胞在effectene转染培养基中生长非常好。

  3. Wnt11r-FLAG上清液的收集:
    1. 在转染培养基中生长4天后,在15ml锥形管中收集培养基。 以1,000rpm旋转5分钟。
    2. 过滤通过硝酸纤维素膜(0.2mm)。根据文献,wnt配体可以在4℃下在培养基中储存几个月而不丧失活性。不清楚如果这是真的为wnt11r。
      可选:以1ml等分试样分装上清液,储存于-80℃。
    3. 通过抗-FLAG珠下拉wnt11r-FLAG蛋白:
      1. 彻底阻止小瓶中的抗FLAG M2凝胶。悬浮液与包装凝胶体积的比例为2:1(即,如果你想得到20μl填充的珠子,使用宽端提取从小瓶中取出40μl)。
      2. 按照制造商的说明清洁防FLAG珠。
      3. 使用冷TBS缓冲液(500μl的40μl的凝胶悬浮液)洗涤珠4次。
      4. 将珠子在5,000-8,000×g离心30秒。在处理样品前等待1-2分钟。用窄端移液管吸头除去上清液。小心不要转移任何 珠。
      5. 用0.1M甘氨酸-HCl pH3.5洗涤珠子以从悬浮液中减少未结合的抗FLAG抗体的痕量
        注意:不要将珠子留在甘氨酸盐酸中超过20分钟。
      6. 用TBS缓冲液再次洗涤珠子共3次。
      7. 向珠子中加入含wnt11r的培养基。 将轮胎上的管子保持在4°C O/N。
        可选:在将蛋白酶抑制剂加入珠子之前,可以向其中加入蛋白酶抑制剂。
        注意:上述每个步骤都应在冰面或4°C进行。
    4. 使用甘氨酸-HCl缓冲液洗脱wnt11r-FLAG蛋白:
      1. 离心珠子和用窄端移液器吸头取出介质。
      2. 用TBS缓冲液洗涤珠子3次。 确保所有的超声波被清除。
      3. 用0.1M甘氨酸-HCl洗脱wnt11r-FLAG蛋白。
      4. 向珠子中加入100μl的0.1M甘氨酸-HCl缓冲液。 孵育样品,在室温轻轻摇动5分钟。
      5. 以5,000-8,000英寸×g离心30秒。
      6. 将上清液转移到含有10μl0.5M Tris-HCl(pH 7.4),1.5M NaCl的新管中。 小心不要转移任何珠子。
      7. 立即使用,将上清液储存在4°C。 储存于-20°C长期储存。
    5. 确定洗脱缓冲液中的蛋白质含量:
      1. 取10μl的洗脱蛋白,加入10μl2×SDS样品缓冲液。 煮沸5分钟,然后进行SDS-PAGE凝胶。 对于标记,在每个泳道中运行不同量的BSA。 用G-蓝染色凝胶,然后确定在10μl洗脱中存在的蛋白质的量。
      2. wnt11r-FLAG的大小为44kDa。

食谱

  1. 媒体
    DMEM
    10%FBS
    1%Pen/Strep

致谢

该协议是在美国宾夕法尼亚大学的Michael Granato实验室开发的,这项工作由NIH授权R01HD037975支持。

参考文献

  1. Jing,L.,Lefebvre,J.L.,Gordon,L.R.and Granato,M。(2009)。 Wnt信号通过斑马鱼拔出/MuSK受体组织突触模式和轴突指导。 < em> Neuron 61(5):721-733。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jing, L. (2012). Cell Culture Transfection for Production and Purification of Wnt Ligands. Bio-protocol Bio101: e176. DOI: 10.21769/BioProtoc.176;
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